Purification and properties of several transfer RNA methyltransferases from S. typhimurium.
A fast method for a single-step fractionation of a number of tRNA methyltransferases from Salmonella typhimurium is described. The method basically consists of ion-exchange chromatography on a phosphocellulose column and permits the separation of the enzymes forming mt6A, m1G, m5U, m7G. The enzyme fractions appear sufficiently purified to allow the estimation of some molecular and kinetic properties. The apparent KM for adenosylmethionine range between 1.5 to 3.2 X 10(-5) M, whereas KM for undermethylated tRNA range between 3.1 X 10(-5) M to 3.1 X 10(-4) M. Glycerol gradient determination indicates the following Mr for the native proteins: 25 X 10(3), 40 X 10(3), 50 X 10(3) and 65 X 10(3) for m7G-, mt6A-, m1G- and m5U-forming enzymes, respectively. A complete analysis of methylated nucleosides formed in vivo in S. typhimurium has been obtained: it also allowed us to infer the pattern of the various tRNA methyltransferases for this prokaryote. The tRNA methyltransferase forming mt6A has been isolated for the first time from any type of cell.
D002852:Chromatography, Ion Exchange; D002855:Chromatography, Thin Layer; D006151:Guanosine / Q000031:analogs & derivatives / Q000378:metabolism; D007700:Kinetics; D008970:Molecular Weight; D011684:Purine Nucleosides / Q000378:metabolism; D012486:Salmonella typhimurium / Q000201:enzymology*; D013912:Threonine / Q000031:analogs & derivatives / Q000378:metabolism; D014529:Uridine / Q000031:analogs & derivatives / Q000378:metabolism; D012359:tRNA Methyltransferases / Q000302:isolation & purification / Q000378:metabolism*
D011684:Purine Nucleosides; D006151:Guanosine; C009182:ribothymidine; C020770:1-methylguanosine; C016578:7-methylguanosine; D013912:Threonine; C030853:N-(N'-methyl-N-(9 beta-D-ribofuranosylpurin-6-yl)carbamyl)threonine; D012359:tRNA Methyltransferases; D014529:Uridine